Metagenomics reveals diversity and abundance of meta-cleavage pathways in microbial communities from soil highly contaminated with jet fuel under air-sparging bioremediation

Abstract

The extradiol dioxygenase diversity of a site highly contaminated with aliphatic and aromatic hydrocarbons under air-sparging treatment was assessed by functional screening of a fosmid library in Escherichia coli with catechol as substrate. The 235 positive clones from inserts of DNA extracted from contaminated soil were equivalent to one extradiol dioxygenase-encoding gene per 3.6 Mb of DNA screened, indicating a strong selection for genes encoding this function. Three subfamilies were identified as being predominant, with 72, 55 and 43 fosmid inserts carrying genes, related to those encoding TbuE of Ralstonia pickettii PK01 (EXDO-D), IpbC of Pseudomonas sp. JR1 (EXDO-K2) or DbtC of Burkholderia sp. DBT1 (EXDO-Dbt), respectively, whereas genes encoding enzymes related to XylE of Pseudomonas putida mt-2 were not observed. Genes encoding oxygenases related to isopropylbenzene dioxygenases were usually colocalized with genes encoding EXDO-K2 dioxygenases. Functional analysis of representative proteins indicated a subcluster of EXDO-D proteins to show exceptional high affinity towards different catecholic substrates. Based on V(max)/K(m) specificity constants, a task-sharing between different extradiol dioxygenases in the community of the contaminated site can be supposed, attaining a complementary and community-balanced catalytic power against diverse catecholic derivatives, as necessary for effective degradation of mixtures of aromatics.

Fig. 1

Evolutionary relationships of extradiol dioxygenases and of α-subunits of Rieske non-haem iron oxygenases found in phagemid metagenomic clones exhibiting catechol meta-cleavage activity. The evolutionary histories were inferred using the Neighbour-Joining method and the p-distance model. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. Phylogenetic analyses were conducted in MEGA4 using partial protein fragments. Bootstrap values above 50% from 100 neighbour-joining trees are indicated to the left of the nodes. The scale bar indicates amino acid differences per site. Proteins analysed biochemically in this study are indicated by arrows. A. Extradiol dioxygenases cluster D (EXDO-D). Sequences of closely related proteins from bacterial isolates or identified in culture independent studies (Kasuga et al., 2007; Suenaga et al., 2007) were included in the analysis and XylE of P. putida mt-2 is shown as an out-group. B. Extradiol dioxygenases related to DbtC (EXDO-Dbt). Sequences of closely related proteins from bacterial isolates or identified in culture independent studies (Sipila et al., 2006) were included in the analysis and BphC of B. xenovorans LB400 is shown as an out-group. C. Extradiol dioxygenases cluster K2 (EXDO-K2). Sequences of closely related proteins from bacterial isolates were included in the analysis and DbfB of Terrabacter sp. DBF63 is shown as an out-group. Proteins that were encoded on a metagenomic insert, which also encodes an isopropylbenzene dioxygenase are shaded in grey. Colocalization of EXDO-K2-encoding genes and of isopropylbenzene dioxygenase (IPBD)-encoding genes is indicated by arrows. D. α-Subunits of Rieske non-haem iron oxygenases related to IPBD. Sequences of closely related proteins from bacterial isolates or identified in culture independent studies (Witzig et al., 2006) were included in the analysis and NahAc of P. putida G7 is shown as an out-group. Proteins that were encoded on a metagenomic insert, which also encodes an EXDO-K2, are shaded in grey.